PROJECT SUMMARY: Melanoma is one of the deadliest forms of cancer and is poorly responsive to standard chemotherapeutics, with 48,000 people dying worldwide each year. The incidence of melanoma has risen dramatically in recent decades. A breadth of work has revealed that stromal cells promote tumor cell motility and metastasis. However, much of our understanding of these interactions - and the therapies that are being developed based on that understanding - is either inferred from end-point assays and fixed tumor sections, or is based on visualizing cells at high resolution in in vitro co-culture models. A major limitation to understanding tumor progression is the lack of genetically tractable in vivo model systems that are amenable to high-resolution imaging. I have overcome this obstacle by visualizing and manipulating tumor cells and their microenvironment directly in a zebrafish xenotransplant model. I have demonstrated that the tumor-macrophage interactions visualized in mammalian systems are recapitulated in great detail in human-to-zebrafish melanoma xenotransplants. With a novel Cre recombinase-based reporter of cytoplasmic transfer, I have shown that sustained macrophage/tumor cell contacts allow for the transfer of cytoplasmic molecules from macrophages to tumor cells, thereby instructing tumor cell dissemination. I propose to use complementary approaches, taking advantage of the strengths of each organism to answer outstanding questions in cancer cell biology. The advantage of using zebrafish is the ease in which genes of interest can be identified and validated in vivo. I will then use a combination of in vitro tissue culture and zebrafish to analyze cellular interactions in real-time. Further, I will use mouse models to test our hypotheses in a mammalian in vivo system. With these approaches in hand, I propose to determine: 1. What cytoplasmic molecules are transferred from macrophages to tumor cells for dissemination? 2. How do macrophages transfer cytoplasmic molecules to tumor cells for dissemination? 3. What additional signals from stromal cells in the microenvironment regulate tumor cell cytoskeletal dynamics and cell biological decision-making? My long term goal is to understand how cells communicate with one another to regulate tumor cell behavior in an effort to develop novel biomarkers, and to identify key proteins and processes to target with therapeutics.